This invention relates to the analysis of various compounds in biological fluids or the like and, more particularly, to novel isocyanates capable of reacting with the compounds of interest to form derivatives receptive to tagging, as by radiolabeling, and to methods of analysis using such tagged derivatives.
For a variety of clinical purposes such as, for example, monitoring dosage schedules, monitoring hormone levels, checking for recent ingestion or following pharmacological dynamics of bioavailability, absorption, degradation or excretion, it is of great advantage to measure the concentration of various drugs or the like to the nanomolar or even picomolar level. As is known, radioimmunoassay can accomplish analyses of this type. To carry out an analysis, an acceptable kit or system must include an antiserum, a standard of the compound to be measured, the radiolabeled derivative of the compound to be measured, a buffering agent or agents and, often, a displacing agent. As is known, the antiserum is produced by bleeding animals which have been immunized by innoculation, for example, with the hapten-protein conjugate (immunogen) corresponding to the compound to be measured (typically termed "antigen").
As is known, in general, the technique of radioimmunoassay measures the competition between radioactively labeled antigen and unlabeled antigen for binding sites on the antibody in the antiserum. By adding to the antiserum known amounts of the antigen to be assayed and a radiolabeled analog, a dose-response curve for bound or free antigen vs. concentration of antigen is constructed. After this immunocalibration has been carried out, unknown concentrations can then be compared to the standard dose-response curve for assay. Crucial to this type assay is the existence of radioactive antigens which compete effectively with non-radioactive antigens. Accordingly, in order to obtain the maximum precision, accuracy, sensitivity, specificity and reproducibility of the assay, purified, well-characterized synthetic radioactive antigens are required. The sensitivity refers to the ability of the assay technique to respond to minimal concentrations of the antigen (viz., the hormone, drug or the like being assayed). Maximal sensitivity is attained when the concentration of free, radiolabeled antigen is negligible, and the concentration of unlabeled antigen approaches zero. When the synthetic, radiolabeled antigen is pure and closely matches in conformation the antigen whose analysis is sought, radioimmunoassay is potentially of the highest sensitivity and specificity.
Synthesizing satisfactory radioactive antigens thus involves certain guidelines. Pure reagents with a minimum concentration of by-products should be utilized. In addition, high-yield, gentle reactions which do not rearrange the antigen or hapten are desired as are reactions which cause only minimal structural alterations of the hapten or antigen. Still further, it is desirable to use derivatizing chemistry that minimizes differences in affinity toward the binding reagent, e.g. -- antibody, of radiotracer and native hapten or antigen so that effective competitive assay is possible.
Tagging of the hapten or antigen being assayed can be achieved, as is known, with .sup.14 C or .sup.3 H. However, analyses involving haptens or antigens tagged by this technique are slow and tedious and can generally only be accomplished by liquid scintillation methods. It is accordingly desirable to radiolabel by tagging with an iodine radioisotope such as, for example, .sup.125 I. However, most compounds which are of interest cannot be labeled by this technique and must accordingly be reacted with an iodine-accepting group. Aromatic rings and some heterocyclic rings, especially those activated for facile substitution, are thus preferred constituents for coupling two compounds to provide derivatives receptive to iodine labeling. For this reason, tyrosine methyl ester and tyramine, both containing an activating phenolic hydroxyl group, have been used to provide derivatives which can later be radiolabeled.
One of the conventional methods previously utilized to prepare tagged steroids involves the initial formation of an intermediate adduct by treatment of the steriod with a chloroformate or succinyl, maloyl, fumaroyl, or phthaloyl ester of anhydride followed by a subsequent reaction with the iodine acceptor. This approach is described in Oliver et al., J. Clinical Investigation, Vol. 47, p. 1035 (1968) wherein 3-0-succinyldigitoxigenintyrosine methyl ester and the corresponding .sup.125 I derivative were prepared for use in the radioimmunoassay of digitoxin in humans. A further approach of this type is shown in U.S. Pat. No. 3,810,866 wherein digitoxigenin is linked to derivatives of tyrosine, 4-hydroxyphenylglycine, 3-hydroxytryptophane, tryptophane and histidine by means of a succinyl, maloyl, fumaroyl or phthaloyl group.
The preparation of such derivatives can take up to several months.
A further technique is described in German application Ser. No. 2,331,922, dated Jan. 10, 1974. In this procedure, there is shown derivatives of 14 different cardiac glycosides which are radiolabeled by first opening the terminal digitoxose ring and oxidizing to the dialdehyde with periodate. The dialdehyde is then coupled with a reagent such as L-tyrosine methyl ester hydrochloride. The resulting adduct is then reduced with sodium borohydride to reduce the remaining hydroxyl groups on the end sugar ring and, incidentally, to saponify the ester to the acid. This adduct can then be radioiodinated on the aromatic ring. This method tends to be cumbersome, subject to by-products at the various intermediate stages and further employs relatively strong reagents.
Yet another technique is shown in South African application No. 73-8312, dated Oct. 16, 1973. This coupling technique may be employed with steroidal ketones such as testosterone by first forming the carboxymethyl oxime. The iodine acceptor such as, for example, tyrosine methyl ester, is then caused to react with a typical carbodiimide such as N,N-dicyclohexylcarbodiimide in the presence of triethylamine in methylene chloride to produce the tyrosine methyl ester amide of the original O-carboxymethyloxime.
It is accordingly an object of the present invention to provide novel isocyanates which may be readily coupled to compounds of clinical interest to provide derivatives which are receptive to tagging, as by radiolabeling.
A further object provides novel carbamate and urea derivatives of compounds of biological, clinical or other interest. A related and more specific object lies in the provision of such derivatives which are receptive to radiolabeling.
A still further object is to provide coupling reagents capable of forming carbamates or ureas useful for joining molecules of clinical interest to substrates of interest such as, for example, proteins, enzymes, polypeptides, glass beads, carbohydrates, plastic articles and the like.
A still further object provides a method of radioassay using as reagents the radiolabeled carbamates or ureas described herein.
Yet another object of this invention lies in the provision of novel coupling agents which form adducts with compounds of clinical interest by a facile, gentle chemical reaction.
Another object is to provide the formation of adducts between the novel coupling agents of this invention and the compound of clinical or other interest which is characterized by synthetic breadth, ease of reaction, procedural simplicity and relative freedom from by-products.
A still further object of this invention provides labeled derivatives which improve the reliability of radioimmunoassay, immunoassay and nonimmume-based techniques utilizing, for example, competitive binding agents.
Other objects and advantages of the present invention will become apparent from the following detailed description and from the sole figure which depicts a typical doseresponse curve obtained utilizing, as the radiomarker, a radioiodinated digoxin derivative of the present invention.
While the invention is susceptible to various modifications and alternative forms, there will herein be described in detail the preferred embodiments. It is to be understood, however, that it is not intended to limit the invention to the specific forms disclosed. On the contrary, it is intended to cover all modifications and alternative forms falling within the spirit and scope of the invention as expressed in the appended claims. For exaple, while the present invention will be described in connection with assays carried out by radioimmunoassay techniques, it should be appreciated that the present invention is equally applicable to use in any type of assay involving similar principles. Still further, while most proteins and many peptides can be radioiodinated without coupling an iodine acceptor thereto, it may be advantageous to employ the novel coupling agents of the present invention to add additional iodine-accepting sites to the protein or peptide to allow for greater specific activity. Additionally, in cases where the strong oxidizing and reducig conditions frequently employed in radioiodination procedures may be detrimental to the structure or properties of the proteins, it may be desirable to pre-radioiodinate the novel coupling agents of the present invention, which then may be subsequently reacted, after purification, if desired, with the protein or peptide under chemically mild conditions. Similar considerations may also apply to the larger entities containing proteins such as viruses, bacteria, cells and the like.